Cyclin based inhibitors of CDK2 and CDK4

ABSTRACT

Structural and functional analysis of peptide inhibitor binding to the cyclin D and cyclin A groove has been investigated and used to design peptides that provide the basis for structure-activity relationships, have improved binding and have potential for development as chemical biology probes, as potential diagnostics and as therapeutics in the treatment of proliferative diseases including cancer and inflammation.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 14/492,753 having a filing date of Sep. 22, 2014,now U.S. Pat. No. 9,376,465 having an issue date of Jul. 28, 2016, whichis a divisional application of U.S. patent application Ser. No.13/940,407, having a filing date of Jul. 12, 2013, now abandoned, whichis a continuation-in-part application claiming priority to U.S. patentapplication Ser. No. 13/851,661 having a filing date of Mar. 27, 2013,which claims filing benefit of U.S. Provisional Patent Application Ser.No. 61/616,154 having a filing date of Mar. 27, 2012, all of which areincorporated herein by reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under RO1 CA131368-O1A2awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 24, 2013, isnamed USC-323_SL.txt and is 16,910 bytes in size.

BACKGROUND

CDKs, the cyclin regulatory subunits and their natural inhibitors, theCDK tumor suppressor proteins (CDKIs), are central to cell cycleregulation and their functions are commonly altered in tumor cells.Deregulation of CDK2 and CDK4 through inactivation of CDKIs such asp16^(INK4a), p21^(WAF1), p27^(KIP1) and p57^(KIP2) may override the G1checkpoint and lead to transformation. CDKs interact with certain cellcycle substrates through the cyclin binding motif (CBM) and form acomplex with the cyclin groove of the G1 and S phase cyclins, a surfacebinding site involving a protein-protein interaction. It has been shownthat CDK isoform and substrate selective inhibition may be achievedthrough the use of peptides that block recruitment of both pRb and E2Fand potently inhibit CDK2/CA kinase activity. Inhibition of CDKs thoughthe cyclin provides an approach to obtain selectivity against otherprotein kinases and inhibit only the G1 and S phase CDKs as only thesecontain a functional cyclin binding groove. In particular, CDKs thatregulate the RNA polymerase-II transcription cycle should be unaffectedby cyclin groove inhibitory (CGI) compounds. Although it has been shownthat cancer cells depend on the RNAPII cycle to express anti-apoptoticgenes and that inhibition of transcriptional CDKs leads to potentanti-tumor agents, it is at the same time likely that this will lead toeffects in normal cells and may be responsible for toxicities observedwith current CDK inhibitors being clinically evaluated.

The cyclin binding motif represents a consensus of the cyclin groovebinding sequences found in many cell cycle and tumor suppressorproteins. CGI peptides in transducible form have been shown to inducecell cycle arrest and selective apoptosis in tumor cells in vitro. Thesepermeabilized peptides also act as anti-tumor agents in that whenadministered directly to a SVT2 mouse tumor model, significant tumorgrowth inhibition was obtained and histological analysis showed thattumors underwent apoptosis.

The ATP competitive CDK inhibitors developed to date are generally nonspecific against the single variants in the CDK family. It is believedthat a major component of the anticancer activity of CDK inhibitors isthrough the transcriptional inhibition of CDK7 and 9. While it has beensuggested that transcriptional CDK inhibition may be beneficial forcancer therapy, it is also probable that this will lead to significanttoxicities. The most selective CDK inhibitor described to date is aCDK4, 6 selective compound, PD0332991 (selective vs. CDK2/proteinkinases (CDK4 IC₅₀, 0.011 μmol/L; Cdk6 IC₅₀, 0.016 μmol/L, no activityagainst 36 other protein kinases) ((IC₅₀-half maximal inhibitoryconcentration) although it has apparently not been tested against thetranscriptional CDKs. Regardless, this compound is a potentantiproliferative agent against retinoblastoma (Rb)-positive tumor cellsand induces a G1 arrest, with concomitant reduction ofphospho-Ser780/Ser795 on pRb. Oral administration to mice bearing theColo-205 human colon carcinoma xenografts resulted in marked tumorregression suggesting that it has significant therapeutic potential andthat targeting CDK4/cyclin D may be a viable strategy. In addition tocyclins A and E, the D-type cyclins also contain a functional cyclingroove and CDK4/cyclin D dependent kinase activities may therefore beblocked by cyclin groove inhibitors.

Further oncology target validation for selective inhibition ofCDK4/cyclin D has been demonstrated using models of breast cancer andwhere it was shown that mice lacking Cyclin D are highly resistant tomammary carcinomas induced by erbB-2 oncogene. Further research into therole of Cyclin D in tumor formation made use of a mutant form whichbinds to CDK4/6 but cannot promote catalytic activity. Thiskinase-defective Cyclin D/CDK complex results in more evidence ofresistance to erbB-2 induced tumorigenesis in mice. Combination of thesetwo studies strongly indicates that Cyclin D1/CDK4 kinase activity isrequired for erbB-2-driven tumorigenesis and therefore confirms thatCyclin D1/CDK4 is a promising oncology target. While there are severalreports of potent and selective inhibitors of the CDK2/cyclin A, Esubstrate recruitment, with both peptidic and peptidomimetic compoundsbeing identified, room for additional inhibitor development exists.Moreover, very little has been reported with respect to eitherinhibitors or on the requirements for binding to the cyclin groove ofCDK4,6/cyclin D1.

Accordingly, what is needed in the art are methods for development ofCDK inhibitors, and in particular CDK/cyclin D and CDK/cyclin Ainhibitors.

SUMMARY

A variety of synthetic CDK/cyclin inhibitors are disclosed. Theinhibitors inhibit interaction of a complex formed between a CDK proteinand a cyclin protein with a substrate of the complex. For example, inone embodiment, the inhibitor can include1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole carboxamide (35DCPT)at the N-terminal, βHomoLeu-NMethylPhe-NH₂ at the C-terminal, and alinking group between the N-terminal and the C-terminal, the linkinggroup comprising arginine or an arginine isostere.

In another embodiment the inhibitor can having the following structure:

wherein

-   -   n1 is 1 or 0,    -   n2 is 1, 2, or 3,    -   the aromatic ring bonded to the (CH₂)_(n2) group can include one        or more halogens on the ring,    -   R is hydrogen or ethyl.

In one embodiment, the CDK/cyclin inhibitor can have the followingstructure:

wherein R1 is methyl or hydrogen and R2 has one of the two followingstructures:

In another embodiment, the inhibitor can have the following structure:

wherein

-   -   n1 is 0 or 1,    -   n2 is 3,    -   R1 is an arginine side chain    -   R2 has one of the following structures:

-   -   R3, R4, and R5 are independently hydrogen or a halogen.

In yet another embodiment, the inhibitor can beN-(5-guanidino-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl)benzamide.

And in another embodiment, the CDK/cyclin inhibitor includes a terminalC-cap having the following structure:

-   -   wherein        -   n is 0 or 1        -   R1, R2, and R3 are independently hydrogen, isobutyl, methyl,            ethyl, or propyl groups.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram illustrating a method as may described hereinthat may be utilized in development of an inhibitor as described herein.FIG. 1 discloses “HAKRRLIF” as SEQ ID NO: 2 and “RLIF” as SEQ ID NO: 52.

FIG. 2A is an alignment of binding site residues of cyclin A2 and cyclinD1. FIG. 2A discloses SEQ ID NOS 65 and 66, respectively, in order ofappearance.

FIG. 2B illustrates an overlay of crystal structures of cyclin D1(marked as D1; 2W96) and cyclin A2 (10 KV) illustrating similarities anddifferences of CBM contacting residues.

FIG. 2C is a ribbon representation of the overlay highlighting thedifferences in the cyclin box helices. Cyclin D1 is shown in thelightest strand and the CGI peptide is marked at either end.

FIG. 3 illustrates a correlation between IC₅₀ and interaction energy forseveral cyclin A-peptide complexes.

FIG. 4 is a comparison of the solvent accessible surface of the cyclingrooves of A2 (FIG. 4A) and D1 (FIG. 4B).

FIG. 5 illustrates a modeled complex of the p27 residues 25-49 withcyclin D1 (2W96) overlaid with SAKRNLFGM (SEQ ID NO: 1).

FIG. 6 illustrates an inhibitor,N-(5-guanidino-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl)benzamide,designed according to disclosed methods.

FIG. 7 illustrates one embodiment of a C-terminal capping group forinhibitors as described herein.

FIG. 8 illustrates three inhibitors designed according to disclosedmethods.

DETAILED DESCRIPTION

The following description and other modifications and variations to thepresent subject matter may be practiced by those of ordinary skill inthe art, without departing from the spirit and scope of the presentdisclosure. In addition, it should be understood that aspects of thevarious embodiments may be interchanged in whole or in part.Furthermore, those of ordinary skill in the art will appreciate that theforegoing description is by way of example only, and is not intended tolimit the disclosure.

In general, disclosed herein is a strategy for inhibition of the cyclindependent kinases in anti-tumor drug discovery. More specifically,inhibition may be afforded through the substrate recruitment site on thecyclin positive regulatory subunit. This approach offers the potentialof generating cell cycle specific CDK inhibitors and reduction of theinhibition of transcription mediated through CDK7 and 9, commonlyobserved with ATP competitive compounds. While highly potent peptide andsmall molecule inhibitors of CDK2/cyclin A, E substrate recruitment havebeen reported, the development of new CDK2/cyclin A inhibitors would beof great benefit. Moreover, little information has been generated on thedeterminants of inhibitor binding to the cyclin groove of theCDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drugtarget and it continues to be widely pursued in the development of newtherapeutics based on cell cycle blockade.

Synthetic inhibitors disclosed herein have been developed frominvestigation of the structural basis for peptide binding to the cyclingroove and examination of the features contributing to potency andselectivity of inhibitors. Synthetic inhibitors of CDK4/cyclin D of pRbphosphorylation are disclosed, examples of which have been synthesized,and their complexes with CDK4/cyclin D1 crystal structures have beengenerated as further described herein. Comparisons of the cyclin groovesof cyclin A2 and D1 are presented and provide insights in thedeterminants for peptide binding and the basis for differential bindingand inhibition.

In addition, a complex structure has been generated in order to modelthe interactions of the CDKI, p27^(KIP1), with cyclin D1. Thisinformation has been used to identify unique aspects of cyclin D1 thathave a significant impact on peptide interaction, and which may beexploited in the design of cyclin groove based CDK inhibitors. Peptidicand non-peptidic compounds have been synthesized in order to explorestructure-activity relationship for binding to the cyclin D groove andthe cyclin A groove which to date has not been carried out in asystematic fashion. Disclosed compounds may be useful as chemicalbiology probes to determine the cellular and anti-tumor effects of CDKinhibitors that are cell cycle specific and do not inhibit thetranscriptional regulatory effects of other cyclin dependent kinases.Furthermore, such compounds may serve as templates for structure-guidedefforts to develop potential therapeutics based on selective inhibitionof CDK4/cyclin D activity and of CDK2/cyclin A activity.

FIG. 1 is a flow diagram illustrating a method for developing aninhibitor as described herein. According to the process, a lead peptideis selected that is a known inhibitor, e.g., a known cyclin A/CDKpeptide inhibitor. By way of example, the octapeptide HAKRRLIF (SEQ IDNO: 2), which is highly selective for cyclin A, can be utilized, asillustrated. The structure activity relationship (SAR) can be determinedfor the lead peptide inhibitor as can be the 3-D structure for theinhibitor in complex with a cyclin D, e.g., a cyclin D1. A peptidicfragment of the lead peptide can be truncated, for instance anN-terminal fragment, and a substitute segment (either a substitutepeptidic fragment or a nonproteinogenic replacement) can then be dockedand scored with regard to affinity of the new fragment ligated inhibitorwith the cyclin D or with a cyclin A. For example, the substitutesegment can be selected via structural analysis of the basis for peptiderecognition with the cyclin A and of the decreased potency found in theSAR with the cyclin D, as described further herein. Reiteration andoptimization of the fragment can be carried out to determine a best fitfragment replacement segment for the truncated peptidic fragment. Theprocess can then be repeated for the remainder of the original peptideinhibitor, e.g., for a central region of the inhibitor and a C-terminalregion of the inhibitor. The combined optimized fragments can then forma new inhibitor for the cyclin/CDK interaction, e.g., the cyclinD/CDK4-substrate interaction, the cyclin E/CDK2 substrate or for thecyclin A/CDK2 substrate interaction.

Common amino acid symbol abbreviations as described below in Table 1 areused throughout this disclosure.

TABLE 1 Amino Acid One letter symbol Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V ValMethodsSolid Phase Peptide Synthesis

Peptides were assembled by using standard solid phase synthesis methodon a Argonaut Quest 210 semi-automated solid phase synthesizer. 10equivalents of the C-terminal amino acid were coupled to Rink resin atthe first place using DIEA (0.082 ml) and HBTU (189.6 mg) in 5 ml DMFfor 1 h. Fmoc of the C-terminus amino acid was removed using 20%piperidine in 5 ml DMF for 10 mins before assembly of 10 equivalents ofthe next amino acid using DIEA (0.082 ml) and HBTU (189.6 mg) in 5 mlDMF. Wash cycles (5*10 ml DMF+5*10 ml DCM) were applied to each step inbetween coupling and deprotection of Fmoc. Upon completion of assembly,side chain protecting groups were removed and peptides were finallycleaved from Rink resin using 90:5:5 mixtures of TFA/H₂O/TIS. Crudepeptides were purified using reverse phase flash chromatography andsemi-preparative reversed-phase HPLC methods. Pure peptides werelyophilized and characterized using mass spectrometry and analyticalHPLC. All peptides contained free amino termini and were C-terminalcarboxamides.

Computational Chemistry

Modeled complexes of peptidic cyclin groove inhibitors bound to eitherCyclin A or Cyclin D were generated as follows: SAKRRLXG (SEQ ID NO: 3)series were modeled from the crystal structure the p107 peptide bound tocyclin A (PDB: 1H28). The HAKRRLIX (SEQ ID NO: 4) series were obtainedby hybridizing the peptide conformation of RRLIF (PDB: 10 KV) (SEQ IDNO: 5) and SAKRRLFG (PDB: 1H28) (SEQ ID NO: 6). The Cyclin A structurein this complex was taken from 10 KV. Cyclin D1/SAKRRLXG (SEQ ID NO: 3)and Cyclin D/HAKRRLIX (SEQ ID NO: 4) were generated in a similar mannerusing cyclin D1 crystal structures (PDB: 2W96) where the peptidicinhibitor bound to cyclin A was superimposed with cyclin D1 and followedby deletion of Cyclin A from further minimization of the complex. Afterapplying the CHARMm forcefield in Discovery Studio 2.5 (Accelrys, SanDiego), the Smart Minimizer algorithm comprised of steepest descent andconjugate gradient and an implicit solvent model of Generalized Bornwith a simple Switching (GBSW) were applied to the complex. In general,all peptide residues were flexible. For cyclin A, all protein residueswere restrained and for cyclin D1, the backbone atoms were fixed andapproximately 300 steps of minimization were required for convergence toan energy minimum. The calculate interaction energy protocol of DS 2.5was used to generate non-bonded energy values between peptidic inhibitorand its associated cyclin. This included calculation of van der Waalsand electrostatic energies to provide an estimation of the affinity ofinhibitors

In Vitro Kinase Assay

CDK2/Cyclin A2 and CDK4/Cyclin D1 kinase assays were performed usingfull-length recombinant CDK2/cyclin A2 and CDK4/Cyclin D1 co-expressedby baculovirus in Sf9 insect cells using an N-terminal GST tag on bothproteins. The kinase assay buffer I consisted of 25 mM MOPS, pH7.2; 12.5mM beta-glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA and 0.25mM of DTT was added prior to use. The [³²P]-ATP Assay cocktail wasprepared in a designated radioactive working area by adding 150 μl of 10mM ATP stock solution, 100 μl [³²P]-ATP (1mCi/100 μl), 5.75 ml of kinaseassay buffer I. 10 mM ATP Stock Solution was prepared by dissolving 55mg of ATP in 10 ml of kinase assay buffer I. Store 200 μl aliquots at−20° C. The substrate used is Rb (773-928) protein with 0.2 mg/mlconcentration. The blank control was set up by adding 10 μl of dilutedactive CDK/Cyclin with 10 μl of distilled H₂O. Otherwise, adding 10 μlof diluted active CDK/Cyclin with 10 μl of 0.2 mg/ml stock solution ofRb (773-928). The reaction was initiated by the addition of 5 μl[³²P]-ATP assay cocktail bringing the final volume up to 25 μl andincubating the mixture in a water bath at 30° C. for 15 minutes. Afterthe incubation period, the reaction was terminated by spotting 20 μl ofthe reaction mixture onto individual pre-cut strips of phophocelluloseP81 paper. The pre-cut P81 strip was air-dried and sequentially washedin a 1% phosphoric acid solution with constant gentle stirring.Radioactivity on the P81 paper was counted in the presence ofscintillation fluid on a scintillation counter. The corrected cpm wasdetermined by subtracting the blank control value for each sample andcalculating the kinase specific activity as follows: Calculation of[P³²]-ATP specific activity (SA) (cpm/pmol) Specific activity (SA)=cpmfor 5 ul [³²P]-ATP/pmoles of ATP (in 5 ul of a 250 uM ATP stocksolution). Kinase Specific Activity (SA) (pmol/min/ug or nmol/min/mg)Corrected cpm from reaction/[(SA of ³²P-ATP in cpm/pmol)*(Reaction timein min)*(Enzyme amount in ug or mg)]*[(Reaction Volume)/(Spot Volume)](SignalChem, Richmond, Canada)

Results

Structural Comparison of Cyclin A2 and D1 Binding Grooves

While numerous experimental structures exist for CDK2/cyclin A2 andother cyclin structures have been solved, for many years CDK4 in complexwith the D type cyclins proved refractory to crystallization. Thestructures for CDK4 in complex with cyclin D1 were recently solvedhowever only in complex with ligands binding to the ATP cleft. This dataprovided the opportunity to gain new insights into the cyclin groove ofthe D cyclins and also to determine the basis for their interactionswith cyclin groove inhibitory (CGI) peptides. At the outset of thisstudy, a limited body of data had been generated for CDK4 inhibitionwhere a series of peptides explored biologically as CDK2/cyclin A, Einhibitors were also characterized in terms of their inhibition ofcyclin D1 mediated substrate recruitment. These results determined thathighly potent peptidic CDK2 inhibitors were in general, significantlyless potent against CDK4.

In order to determine the structural and functional differences of thesecompounds, their interactions with the cyclin D1 recruitment site weremodeled and compared with known cyclin A complex structures. In terms ofcyclin A binding, optimized peptides (i.e. the octamer, HAKRRLIF, p21sequence (SEQ ID NO: 2)) contain three major determinants which arerequired for high affinity binding. As illustrated in FIG. 2, theseinclude a primary hydrophobic pocket which interacts predominantly withleucine and phenylalanine residues of the peptide, an acidic regionwhich forms ionic contacts with basic peptide residues and a secondaryhydrophobic pocket occupied by either an alanine or valine of the cyclinbinding motif (CBM). While the majority of CGI peptide contactingresidues are identical or semi-conserved in both cyclin isotypes, twonotable exceptions were observed. In cyclin D1, Val60 (interacts withPhe8) and Thr62 (close to Arg4) are substituted for Leu214 and Asp216 incyclin A2 respectively. As these residues in the cyclin A context, makecontacts with major determinants of cyclin A binding, it is expectedthat even semi-conservative replacements would lead to significanteffects on cyclin groove inhibition.

Upon overlay of the corresponding alpha carbons of the two cyclins,other semi-conserved and non-conservative differences were observed inthe structural comparison. These residues are not as significant forbinding of the octapeptide however their proximity to the cyclin bindinggroove suggests that they have potential for exploiting in the design ofselective CDK inhibitors targeting cyclin D1. Of the non peptidecontacting residues, the largest structural variation is in the exchangeof Y286 of cyclin A for I132 of cyclin D1. Overlay and comparison of theC-alpha trace of the two structures indicates that this variation,coupled with the relative movement of a helix-loop segment (residues119-136 of 2W96) leads to a significant conformation variation proximalto the cyclin groove. This region as a consequence is considerably moreopen in cyclin D1 and provides an extension to the primary hydrophobicpocket. This additional pocket could therefore accommodate larger ligandgroups than would be feasible for cyclin A inhibitors.

Sequence alignment of binding sites for cyclin A2 (top) and cyclin D1(bottom) are shown in FIG. 2A. Residues that contribute to selectivityare shown in italics. The alignment reveals that while a majority of theresidues are conserved, Leu214/Val60, Asp216/Thr62, Glu224/Glu70, andArg250/Lys96 are the main residues that are responsible for selectivity.

FIG. 2B illustrates an overlay of crystal structures of cyclin D1 (2W96)and cyclin A2 (10 KV) illustrating similarities and differences of CBMcontacting residues. The Leu and Phe residues of the CBM interactingwith the primary hydrophobic pocket are shown at 100. E220 and D216comprise the acidic region and the secondary hydrophobic pocket is tothe left of W217.

FIG. 2C is a ribbon representation of the overlay highlighting thedifferences in the cyclin box helices. Cyclin D1 is shown as the lightribbon and the ends of the CGI peptide are marked. The region displayingthe largest structural differences after superimposing the backboneatoms is marked between 101 and 102 (residues 116-136 of cyclin D1).

Another consequence of the differing conformation and composition of the116-136 region affects the secondary hydrophobic pocket with which theCGI peptide Ala2 interacts. I281 of cyclin A2 is a Tyrosine residue(Y127) in D1. The kinked helix containing this residue is shiftedtowards the groove, bringing this residue closer to the peptide anddecreasing the volume of the lipophilic pocket on the peptide N-terminalside of the W63 (FIG. 2B).

Structural Basis for Cyclin D1 Inhibition

Prior to detailed analysis of modeled peptide-cyclin D1 complexes, thestructural and energetic basis for potencies of cyclin A inhibitors wasexamined. Since a complete set of cyclin A crystal structures forpeptides with cyclin D1 affinity is not available, a cyclin A complexfor HAKRRLIF (SEQ ID NO: 2) was first constructed. This peptide ishighly selective for cyclin A versus cyclin D1. Formation was completedby building on existing pentapeptide (10 KV) and octapeptide structuresto supplement those available for PVKRRLDL (E2F) (SEQ ID NO: 7) andSAKRRLFG (p107) (SEQ ID NO: 6) CBM sequences. The non-bondedinteractions of these crystallographic complexes were estimated bycalculation of per residue and total interaction energy values (DS 2.5,Accelrys) to determine individual contributions and to establish ifthese were reflective of the observed affinities (approximated byinhibition constants). These values shown in Table 2, below, delineateda relationship in terms of both previous SAR of individual residues andCGI potency.

TABLE 2 (SEQ ID NOS 2, 6, 7, 5, 8, 2, 6, 7, 5 and 8, respectively, inorder of appearance) Cyclin A Cyclin A Cyclin A Cyclin A Cyclin A H−65.1 S −63.9 P −23.1 A −18.0 A −19.2 V −15.8 K −42.2 K −40.6 K −47.4 R−72.3 R −69.7 R −74.3 R −111.3 Cit −38.4 R −58.7 R −25.2 R −9.3 R −46.4R −47.2 L −11.7 L −12.8 L −9.9 L −13.8 L −13.8 I −6.8 F −12.2 D 0.6 I−0.1 I −0.06 F −23.5 G −4.2 L −15.4 F −19.5 F −19.5 Total −298.3 −247.8−194.6 −191.1 −119.06 Cyclin D Cyclin D Cyclin D Cyclin D Cyclin D H −20S −24.1 P −18.8 A −6.3 A −5.6 V −11.9 K −44.6 K −44.7 K −52.2 R −54.7 R−57 R −47.6 R −106.9 Cit −30.3 R −27 R −17.6 R −11.1 R −19.2 R −19.2 L−15.2 L −13.7 L −14.7 L −14.1 L −14.1 I 0.7 F −10.2 D −1.6 I 0.2 I 0.2 F−13 G −4.7 L −11.7 F −13.9 F −13.9 Total −180.1 −177.6 −169.6 −153.9−77.3

As determined through sensitivity to major potency loss by alaninesubstitution and other residue replacement, as shown, the energeticanalysis shows the critical Arg4 of the octapeptide makes an extensivecontribution to binding, whereas that of the less sensitive Arg5 islower. Truncation of the His-Ala-Lys N-terminal sequence has beenpreviously shown to result in a decreased affinity for cyclin A with thepotency decreasing approximately 100 fold. The contribution of thesethree residues to binding is confirmed through the energetic analysiswhere His1 and Lys3 especially provide favorable interactions with thebinding pocket. The total binding energies of both HAKRRLIF (SEQ ID NO:2) and RRLIF (SEQ ID NO: 5) calculated (−298 vs −188) correlate wellwith the inhibition constants of these two compounds. Further analysisof the cyclin residue energetics determined that acidic residues,including Asp216, Glu220, Glu224 and Asp283 allow favorableelectrostatic contacts with the basic peptide N-terminal sequence. Inaddition, the energetics of the contribution of Ala2 to bindingcorrelates well with observed potency increase of the Ser-Ala mutationin the p21 C-terminal context.

Further correlation of the interactions and contributions of theC-terminal sequence of the CBM interacting with the primary hydrophobicpocket (FIG. 2A) in addition to visual inspection of the non-bondedcontacts in the p21, p107 and E2F contexts, indicates the structural andenergetic differences. In varying peptide sequence contexts, the p21Leu-Ile-Phe (LIF ‘motifette’) sequence has been demonstrated to be morepotent than the p107 (and p27) LFG and E2F, LDL motifettes. Table 2illustrates that while the Leucine contributions in each context aresimilar, the Phe side chain provides increased complementarity in thep21 sequence (−23.5 kcal/mol vs. −12.2) resulting in its 2-3 foldgreater affinity compared to the LFG sequence. More favorable contactsare observed due to the geometrical arrangement of the aromatic sidechain allowed by the spacer residue between the Leu and Phe in the p21context. Overall, the energetic analysis of peptide binding to cyclin Aconfirms that a relationship exists between calculated binding enthalpyand experimental affinity and additionally that individual residueenergetics closely correlate with the SAR and contribution of CBMdeterminants. This relationship provides the basis to perform ananalysis of peptide binding to cyclin D1 and to determine the structuralbasis for decreased affinity of cyclin D1 inhibitors and therefore tofacilitate the design of more potent compounds.

The intermolecular complexes of cyclin D1 with the above peptides wereformed by superposition of the apo-cyclin D1 structure (2W96) with thecrystallographically derived cyclin A bound structure of the CBMcontaining peptides and followed by deletion of cyclin A. Theenergy-minimized structure was then calculated using the CHARMmmolecular forcefield, and the similarities and differences of cyclinbinding motif−cyclin interactions were examined.

In order to further probe the molecular consequences of variations inbinding residues, the intermolecular energies were calculated for theinteractions of each of the peptides with cyclins A and D1. In line withthe observed potencies of each compound and selectivity for cyclin A, acorrelation was determined between affinity (kinase inhibition) andtotal interaction energy (CIE) calculated for 4 peptides ranging in IC₅₀from 0.021 to 99 μM. Results are illustrated in Table 3, below and FIG.3.

TABLE 3 SEQ Interaction Energy IC50 Cyclin A ID NO: (Kcal/Mol) (μM)LogIC50 HAKRRLIF 2 −298.3 0.021 −1.68 SAKRRLFG 6 −247.8 0.073 −1.14PVKRRLDL 7 −194.6 1.2 0.08 RRLIF 5 −191.1 7.7 0.89

For this relationship, an R² of 0.91 indicated that the both the crystaland modeled structural complexes were accurate and that the establishedcorrelation is useful as a predictive tool for design and synthesis ofmore potent and selective compounds. Comparison of the predictedaffinities of each peptide also demonstrated that the CIE correlateswell with the selectivity of the compound for cyclin A (Table 2). Thiswas additionally confirmed by a second method for estimation of bindingaffinity. Calculation of Ludi Scores provided results directly in linewith the relative potencies on A vs. D1. Further analysis of theindividual energetic contributions of residues of both the peptide andcyclin in each context revealed further evidence for the structuralbasis of CGI selectivity. Not surprisingly, it was observed that thecyclin D binding site variations described above contributed extensivelyto the selectivity of each peptide for cyclin A2. Of course, anyinhibitory peptide may be utilized in a modeling process as disclosedherein. In general, the peptide inhibitor will be relatively short, forinstance about 10 amino acids or less in length, or about 8 amino acidsor less in length, such as the octapeptides, pentapeptides, andtetrapeptides specifically detailed herein.

The optimized p21 derived peptide, HAKRRLIF (SEQ ID NO: 2) is highlyselective for A (0.021 μM) vs. D1 (6 μM). In addition to the totalinteraction energy describing the non-bonded interactions of thepeptide-cyclin interaction, the individual contributions of residuesfrom both molecules was determined. These results indicate that thehighly basic N-terminal residues interact much more favorably with thecyclin A groove. As no crystal structure is available for this peptide,an A complex was modeled on the basis of the residue contacts of RRLIF(10 KV) (SEQ ID NO: 5) and SAKRRLFG (1H28) (SEQ ID NO: 6). Analysis ofprotein-peptide contacts and interaction energies reveals that a greaterconcentration of acidic residues in A2 compared to D1 contributesextensively to this selectivity. In particular Asp216 of cyclin A2(which is aligned with T62 of cyclin D1) provides a favorable additionof 17 kcal/mol to the binding energy in interactions with Arg4. Thiscontribution is largely absent in the cyclin D1 complexes modeled wherethe hydroxyl group of T62 weakly interacts with Arg4. When theinteraction of both Arg4 and Arg5 are considered, the calculated bindingenergy of these two residues for cyclin A is more than twice thatobserved for cyclin D1. Glu220 in Cyclin A2 interacts with Arg4similarly to the corresponding residue (Glu66) in Cyclin D suggestingthat the energetic differences are mainly due to the absence of thesecond acidic residue in D.

As mentioned above, comparison of the CGI peptide binding residues incyclin D1 revealed that a valine residue occupied the position observedas a leucine in A2 (Leu214Val). As this residue is located in thelipophilic pocket interacting with the LIF motif of p21, the immediateconclusion is that this contributes significantly to peptide selectivityfor A vs D. Initially, this appears to be counterintuitive since valineis a smaller residue and might be expected to provide a larger bindingpocket. Close examination of the position of Val60 indicates that theshorter and less flexible side chain brings the interacting methylgroups closer to the phenylalanine of the peptide and thereforedecreases the volume of the hydrophobic pocket (FIGS. 2B, 4B). This wasconfirmed upon overlay of cyclin A2 bound to HAKRRLIF (SEQ ID NO: 2)with the cyclin D1 modeled complex, where a significant steric clashwith the Phe8 side chain was observed (FIGS. 2A, 2B). This suggests thatthe binding mode of Phe8 with cyclin A2 is not compatible forinteraction with cyclin D. In order to determine the consequences of theoverlap, the complex formed between cyclin D1 and HAKRRLIF (SEQ ID NO:2) was subjected to energy minimization to relieve this overlap. Asignificant displacement of the phenylalanine was observed and which didnot come at the expense of Leu6 (peptide residue), whose position wasnot affected. Further analysis of the interaction energy and comparisonwith the values calculated for octapeptide inhibition of both cyclins,indicated a reasonable correlation between predicted and calculatedper-residue affinity of the C-terminal motifette. These data suggestthat displacement of the aromatic side chain comes at the expense of itscomplementarity with the primary hydrophobic pocket and that the valinesubstitution is responsible for the significant decrease in affinity forcyclin D1.

FIG. 4 is a comparison of the solvent accessible surface of the cyclingrooves of A2 (FIG. 4A) and D1 (FIG. 4B). The individual subsites of theCBG are labeled for each cyclin. Examination of the intermolecularcontacts and interaction energies for SAKRRLFG (p107 cyclin bindingmotif) (SEQ ID NO: 6) with cyclin D1 reveals a similar pattern ofresidue energetics for the basic region of the peptide as in theHAKRRLIF (SEQ ID NO: 2) context. SAKRRLFG (SEQ ID NO: 6) has a loweraffinity for cyclin A, with the less optimal geometry of the LFGmotifette resulting in a reduced contact surface area of the phenyl ringwith the pocket. Calculation of the individual residue interactionenergies suggests that the presence of Val60 has a markedly smallerimpact on affinity of the p107 peptide for cyclin D1 than in the p21context due to the different approach angle of the interacting sidechain, and that the selectivity results from increased affinity of theArg4Arg5 determinant with cyclin A2.

Comparison of the E2F CBM, PVKRRLDL (SEQ ID NO: 7) (Table 3, FIG. 4)reveals further insights into the structural basis for CGI selectivityfor cyclin A and after comparison of the binding energetics againindicates less favorable contacts with the peptide in the cyclin D1context (Table 2). As has been previously described, the LDL containinginhibitors generally have a decreased binding relative to the LIFcompounds and in this case is reflected in the 50 fold increased IC50value. In contrast to the LFG sequence, the LDL sequence has asubstantially lower predicted affinity for hydrophobic pocket of cyclinD1, consistent with the observed inhibition constants.

Further Analysis of Peptide SAR and Insights into the Design ofSelective Cyclin D1-CDK4 Inhibitors

The insights into the structural basis for peptide recognition forcyclin A and for the decreased potency against cyclin D1, providedfurther opportunity to expand inhibitor structure activity relationshipsby including additional derivatives. As suggested from the abovestructural analysis, differences in the primary hydrophobic pocket werethe major determinants in cyclin A selectivity of the studied peptides.These observations predicted that analogs with variant C-terminal groupsmay interact with the cyclin D pocket with differing affinity than tothe cyclin A groove. Based on this observation, further peptides weredesigned to exploit these structural differences and generate compoundswith increased affinity for cyclin D1. Due the decreased volume of theprimary pocket in cyclin D1, a series of non-proteinogenic cyclicreplacements for Phe7 (p107) and Phe8 (p21) cyclin binding motifcontaining octapeptides were designed. A series of 5 and 6 membered ringsystems were incorporated into the p21 (HAKRRLIX (SEQ ID NO: 4)) andp107 (SAKRRLXG (SEQ ID NO: 3)) contexts (Table 4, below). As shown,these included 2-furylalanine (X1), 2-thienyl alanine (X2),3-thienylalanine (X3), cyclobutylalanine (X4), cyclopentylalanine (X5),cyclohexylalanine (X6) and 3 and 4 pyridyl alanine residues (X7 and X8)providing for the most part isosteric functionalities mimicking theinteractions of the phenylalanine.

The inhibition of CDK activity was determined through a standard filtercapture assay involving a GST-labeled Rb protein and quantification ofthe incorporation of 32P into the substrate. Activities of peptidespreviously tested against CDK2A and CDK4D were determined using thisassay format. Although similar constructs and substrate was used,significant differences in potency were observed. In particular the IC50for HAKRRLIF (SEQ ID NO: 2) was approximately 10 fold higher thanpreviously determined (1.3 vs. 0.14 μM) and the inhibition of CDK4/D1was more pronounced than before (1.6 vs. 6 μM). These differences may beaccounted for in slight differences in amount of cyclin in the proteinprep and excess cyclin or CDK would result in data variation. As aconsequence, it was decided that structure-activity relationshipsdetermined using the kinase assay were best interpreted by functionalcomparisons calculated relative to the native p21 or p107 sequence ineach assay. Data is therefore presented as a ratio of each C-terminaland other analogs activity in addition to the IC50s presented for eachcompound. Results are shown in Table 4, below.

TABLE 4 SEQ IC50 Po- IC50 Po- IC50 ID CDK2/ tency CDK4/ tency CDK2/SEQUENCE NO: A2(μM) ratio D1(μM) ratio E(μM) p107 SAKRRLFG 6 3.3 2.9SAKRRLX1G 9 9.1 2.8 7.5 2.6 4 SAKRRLX2G 10 27 8.2 11.4 3.9 SAKRRLX3G 111 0.3 6 2.1 SAKRRLX4G 12 100 30.3 74 25.5 SAKRRLX5G 13 18 5.5 28 9.7SAKRRLX6G 14 83 25.2 36 12.4 SAKRRLX7G 15 80 24.2 51 17.6 SAKRRLX8G 16750 227.3 143 49.3 HAKRRLIF 2 1.3 1.5 0.3 p21 HAKRRLIX1 17 6.1 4.7 11.47.6 1.3 HAKRRLIX2 18 3.6 2.8 6.5 4.3 HAKRRLIX3 19 25 19.2 100 66.7HAKRRLIX4 20 25 19.2 100 66.7 HAKRRLIX5 21 20 15.4 90 60.0 HAKRRLIX6 2258 44.6 6.3 4.2 HAKRRLIX7 23 29 22.3 28 18.7

For the 2-furylalanine replacement (X1) in the p107 context, it wasfound that kinase activity induced by this compound decreased a similaramount in both CDK2A (2.8 fold) and CDK4D (2.6 fold) although slightlyless so for the latter. In the p21 context more of a differential wasobserved (4.7 and 7.6 fold decrease respectively). The p107 X2derivative (2-thienylalanine) data indicates that the potency decreaseagainst cyclin D was considerably reduced (3.9 fold) relative to cyclinA (8.2 fold decrease). A similar differential was observed for the p21X2 derivative (2.8 vs. 4.3 fold respectively). The X3 amino acid,3-thienylalanine was found to be less potent than X2 in both p107 andp21 contexts.

Examination of the results for aliphatic cyclic amino acid replacements,including cyclobutyl (X4), cyclopentyl (X5) and cyclohexylalanine (X6),indicated that depending on the CBM context, different selectivityprofiles were observed. X4 resulted in dramatic potency decreases inboth contexts however significantly more so with cyclin A. Both the p21and p107 versions incorporating X5, indicate that it is tolerated to alarger degree in binding to cyclin A. Conversely, the p21 derivative ofX6 is tolerated to a significantly larger degree in binding to cyclin D1with only a 4 fold drop-off observed compared to 45 fold withCDK2/cyclin A. If the IC50s of this compound are considered, it issignificantly more potent towards CDK4/cyclin D1 than againstCDK2/cyclin A (6.3 vs. 58 μM). A similar trend was shown for the p107 X6sequence although was not as dramatic. An interesting set of results wasobtained for the pyridylalanine derivatives where one carbon of thenative Phe residue is replaced with nitrogen. A large decrease inactivity was observed for these compounds in both p21 and p107 variants.Binding to cyclin D1 for these analogs was again tolerated to a largerdegree, especially with the 3-pyridylalanine derivative (X7) in the p107context. Unexpectedly, the activity of 4-pyridylalanine (X8)incorporated in SAKRRLXG (SEQ ID NO: 16) decreases 200 fold relative tothe native sequence in terms of cyclin A but 46 fold in the p21 X8derivative. Further analysis of the p21 analog binding to cyclin D1indicates that the X8 containing peptide loses all activity towardsCDK4/cyclin D1. The binding of X7 to cyclin D1 decreases 17.6 foldrelative to the phenylalanine in the LXG motif and 18.7 fold in the LIXcontext.

Structure-Activity Relationship for Peptide Binding to Cyclin D1

For the CDK4/cyclin D1/pRb SAR of the Phe replacements in the SAKRRLXG(SEQ ID NO: 3) context, the most potent analog is the furylalanine, X1derivative with an IC50 of 7.5 μM with X2, the 2-thiophene containingpeptide being slightly less potent (11.4 μM). The order of potency isreversed in the p21 CBM since HAKRRLIX2 peptide (SEQ ID NO: 18) hasapproximately 2 fold greater inhibition than the furylalanine containingpeptide (6.5 and 11.4 μM respectively). The 3-thienyl analog X3undergoes a potency drop off relative to X2 in both contexts.Cyclobutylalanine incorporation into the p107 context retained a levelof binding as do HAKRRLIX5 (SEQ ID NO: 21) and SAKRRLX5G (SEQ ID NO: 13)although this is weak relative to the native sequences. Thecyclohexylalanine replacement, X6 was of equivalent potency to thethiophene containing peptide in the HAKRRLIX (SEQ ID NO: 22) context,however of notably higher inhibition than the p107 derivative (6.3 μM vs36 μM). The 3-pyridylalanine peptides (X7) were considerably moresignificant inhibitors when incorporated C-terminal to the Ilecontaining spacer residue and which has previously been shown to allowmore favorable geometry for binding. The 4-substituted derivative (X8)are weaker binders in both CBM contexts however with 143 μM IC50observed in the CDK4/cyclin D1 kinase assay for SAKRRLX8G (SEQ ID NO:16) and no observable activity for HAKRRLIX8 (SEQ ID NO: 24). For themost part, the p21 sequences follow the previously observed trend asbeing more potent than the p27 and p107 peptides. Two C-terminal analogshowever have higher affinity when incorporated with the p107 residues,these being the furylalanine (X1) and 4-pyridylalanine (X8) containingpeptides.

Additional insights into cyclin groove interactions in cyclin D1 areprovided by C-terminal and other derivatives incorporated into HAKRRLIF(SEQ ID NO: 2). The p-fluorophenylalanine (4FPhe) derivative has beenpreviously shown to significantly increase the inhibitory potential ofpeptide cyclin A inhibitors with respect to the native residue. Incontrast to these results, synthesis and testing of RRLI(4FPhe) (SEQ IDNO: 25) resulted in decreased inhibition of CDK4/cyclin D1 kinaseactivity (compared to HAKRRLIF (SEQ ID NO: 2), a 160 fold decrease) vs.only a 20 fold decease in CDK2/cylin A activity).

As discussed in above sections, there are differences in the Arg4interacting residues in cyclin D1 vs. cyclin A2 and that thesevariations contribute to decreased binding of peptides to cyclin D1.Specifically, cyclin A has two acidic residues that interact with thepositively charged side chain compared to only one in cyclin D1. Thisresidue has previously been shown to be critical for cyclin A bindingactivity. It would therefore be predicted that replacement of the Argwith an isosteric residue would have less of an impact on cyclin Dbinding. Incorporation of citrullene into p21 to generate peptide,HAKCitRLIF (SEQ ID NO: 26) in order to determine effect on inhibition ofcyclin D confirmed that Arg4 is significant for interaction with cyclinD1, as shown in Table 5. The ratio the activities of the Cit and Argcontaining peptides in both contexts revealed that its effect on cyclinD1 activity (14 fold potency decrease) was similar to that observed incyclin A. This result was corroborated by comparison of the activitiesof citrullene incorporated into pentapeptide, RCitLIF (SEQ ID NO: 27).Compared to the octapeptide sequence, the 5mer potency decreased roughly120 fold for cyclin A (1.3 vs. 164 μM) and cyclin D1 (1.5 vs. 179 μM).

TABLE 5 SEQ IC50 ID IC50 Potency CDK4/ Potency SEQUENCE NO: CDK2/A2(μM)ratio D1(μM) ratio SAKRRLFG 6 3.3 — 2.9 — HAKRRLIF 2 1.3 — 1.5 — RRLIpfF25 26 20.0 250 166.7 HAKCitRLIF 26 18 13.8 21 14.0 HAKTRLIF 28 50 38.525 16.7 CitRLIF 8 164 126.2  179 119.3 SCCP10 25 19.2 8 5.3 SCCP5624 >100 — 60 20.7 SAKRNLFGM 1 — — 146 — SAKRNLFG 29 — — 75 — SAKRALFGM30 — — 68 — PAKRRLFG 31 8 — 6.7 — PVKRRLFG 32 3 — 28 — PVKRRL3CFG 33 1 —3.2 —

Inhibitors are described in Table 6, below. In Table 6, 3TA is3-thienylalanine, bLeu is betahomoleucine, CHA is cyclohexylalanine, anddimethyllysine is lysine with the epsolon amino group methylated.

TABLE 6 SCCP SEQ ID CDK2/cyclin A IC50 CDK4/cyclin ID Peptide SequenceNO: (mM) D1 IC50 (mM)  540 RRLNpfF 34 0.58    8 5811 RRLIF 5  1.4 ± 0.4216.1 ± 1.73  5812 Cit-RLIF 8 23.7 ± 8.49 72.3 ± 11.09 5831 RCitLIF 27 6.4 ± 2.76 46.5 ± 17.04 5832 RPLIF 35 ~100      >180 5833 RALIF 36 11.3± 3.54 >100 5871 RRLFG 37 19.4 ± 1.77 >100 5873 RGLIF 38  87.0 ±30.05 >180 5874 RRLF 39  24.8 ± 12.66 100~180  5875 RR{bLeu}F 40  3.3 ±2.33 20.7 ± 12.45 5876 RR{bLeu}FG 41  2.8 ± 0.78 22.4 ± 14.57 5877 RXLIFX is DMAM 42 >>100       >>180  peptoid 5878NNC11-X-LIF >>100       >>180  DMAM peptoid 5879 RZLIF Z = DMAMAla43 >>100       >>180  peptoid  457 SAKRRLFG-NH2 44 0.30 ± 0.15 1.6 ±0.55 5815 SAKRRLFG-OH 45 0.35 ± 0.15 1.2 ± 0.38 5814 SAKRRL3TA G-OH 460.43 ± 0.23 3.9 ± 0.55 5820 SAKRR{bLeu}FG- 47  0.13 ± 0.014 0.48 ± 0.090OH 5813 SAKRR{bLeu}3TAG- 48 0.31 ± 0.25 0.59 ± 0.015 OH 5816HAKRRLI{CHA} 22 0.25 ± 0.21 0.26 ± 0.13   444 HAKRRLIF 2  0.13 ± 0.0350.22 ± 0.11  5941 R{bLeu}NMeF-NH2 0.405 ± 0.091     89.65 5925R(NMeArg)LIF 49 13.9   5930 R{bLeu}NMeF 0.505 ± 0.36      61.17 5918R(dimethyllys)LIF 50 5.4     72.2

Insights into SAR for interaction of cyclin D1 inhibitors of thesecondary hydrophobic was revealed through synthesis of peptidescontaining the E2F and p107 CBMs. A preference for smaller side chainswas indicated by the increased inhibition of PAKRRLFG (SEQ ID NO: 31)compared to that of PVKRRLFG (SEQ ID NO: 32). This result is inagreement with the structural analysis which shows a decreased volume ofthis subsite in cyclin D1 compared to A

From previous studies into the replacement of peptide determinants withfragment alternatives, compounds were identified where the p21 LIF motifwas replaced with a Leu-bis-aryl ether system, while maintaining asimilar potency level for cyclin A2 inhibition. A compound wassynthesized incorporating 3-phenoxybenzylamide replacing the Phe andalso N-terminally capped with1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carboxylic acid andsubsequently tested for inhibition of CDK4/D1 (SCCP10 on Table 5).SCCP10 was found to have a respectable inhibition of cyclin D1 and inaddition, its relative potency compared to the p21 octapeptide wasenhanced compared to cyclin A inhibition. SCCP10 is 5 fold less potentthan HAKRRLIF (SEQ ID NO: 2) towards cyclin D1, however undergoes a 20fold drop off when cyclin A2 activity is considered. A similar trend wasobserved for the SAKRRL-3PBA peptide (SEQ ID NO: 51) small moleculehybrid 3-phenoxybenzylamide end capped peptide when tested against bothcyclin grooves although in this context the cyclin A differential wasnot as profound. Arg-Arg-13-homoleucyl-3-phenoxybenzylamid (SCCP 5624)was also synthesized and shown to be selective for CDK4/cyclin D1.

The Phe side chain of the octopeptide HAKRRLIF (SEQ ID NO: 2) wasreplaced with smaller side chains in a series of compounds as shownbelow in Table 6. SCCP396, possessing furyl-Ala replacement was indeedselective for cyclin D1 (15% of kinase activity enhancement for cyclinD1 vs. A2). Other replacements with larger ring systems (SCCP 397, 401,402) were not as favorable. The smaller side chains thus reacted morefavorably with cyclin D1.

N-Terminal Partial Ligand Alternatives:

Derivatives and Isosteres of 1-phenyl-1H-1,2,4-triazole-3 carboxamide.

Based upon the above results and other known compounds (see, e.g.,Andrews, et al. ChemBioChem, 2006, 7, 1909-1915), the N-terminalArginine of the p21 RLIF tetrapeptide was substituted with a series ofdifferent heterocyclic isosteres capable of interactions similar tocritical amino acids of the parent peptide and the triazole. Pyrazole,furan, pyrrole and thiazole systems were synthesized and varioussubstitutions of the phenyl ring were explored. The N-caps were ligatedto the tetra peptide using solid phase synthesis, purified by reversephase HPLC and characterized by MS.

In vitro binding and functional assays were performed in order to studythe inhibitory effect of compounds on CDK2/Cyclin A prior to furtherevaluation in cell viability assays to determine antitumor effects. Onthe basis of the results, further high throughput docking of potentialheterocyclic fragments was carried out to identify N-capping groups ofvarying chemical diversity for synthesis and in vitro testing.

A phenyl 1,2,4-triazole series (Scaffold I) was utilized as a basis fordevelopment of a family of phenylheterocylcic compounds as potentialN-capping groups for cyclin A and/or cyclin D inhibitors. The generalstructure of the compounds was:

wherein R₁, R₂, R₃, R₄ are independently hydrogen, halogen, methyl, ormethoxy and W, X, Y, and Z are independently C, N, O, or S.

Compounds containing the 1-phenyl-1H-pyrazole-3-carboxamide substructurewere synthesized as a potential scaffold and are described in Table 7,below. The synthesis was achieved through a scheme in whichethylacetopyruvate was condensed with the corresponding substitutedphenyl hydrazine. Initial attempts involved base catalysis of thereaction upon which two isomers were obtained. The desired isomer wasidentified and confirmed through 1-D NOE analysis where irradiation ofthe R4 methyl group led to an enhancement of the two ortho aromatichydrogens. This reaction was further optimized by performing thecyclization in acidic conditions thereby protonating the hydrazine andsuppressing formation of the non-desired regioisomer. The versatilepyrazole synthesis allowed generation of a variety of analogs includingthe unsubstituted phenyl, the 3-methoxy and 4-methoxy phenyl as well asthe 3,5 dichloro, 3 chloro and the 4 chlorophenyl compounds.

The triazole core structure of1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carbonyl wasreplaced with isosteres such as pyrazole, furoic acid, pyrrole, andthiazole appropriately substituted with a carboxylic acid group and aphenyl ring. Multiple capping groups were synthesized and ligated withthe tetra peptide RLIF (SEQ ID NO: 52). The synthetic schemes forpyrazoles, furan and pyrroles are outlined in scheme1a, 1b and 1crespectively.

The X-ray crystal structure of(1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carbonyl-RLIF (SEQID NO: 53) shows that the N-cap hydrogen bonds with Trp217 and Gln 254of cyclin A. SAR information in Table 6, below reveals the following:

-   -   Triazole N-caps were found to be the most potent of the tested        compounds, followed by pyrazole and furan. The 4-chloro        substitutions on the phenyl ring are the most effective,        followed by 3,5-dichloro substituted compounds.    -   Pyrrole and thiazole show significantly lower activity than the        triazole Ncaps.

TABLE 7 CDK2/ CDK4/ SCCP cyclin cyclin ID A IC₅₀ D1 IC₅₀ No. R₁ R₂ R₃ R₄W X Y Z (mM) (mM) Triazole 5843 H H H H N N N C 16.2 ± 3  48.7 5773 Cl HCl CH₃ N N N C   4 ± 0.6 27 5774 H Cl H CH₃ N N N C 11.5 ± 3.3 11.3Pyrazole 5762 H H H CH₃ N N C C 40.3 ± 6.5 53.8 5763 Cl H Cl CH₃ N N C C 21.8 ± 13.7 100~180 5764 Cl H H CH₃ N N C C 11.9 ± 2.0 45 5771 F H HCH₃ N N C C  29.6 ± 12.2 69.6 5765 H Cl H CH₃ N N C C 33.7 ± 8.1 49 5766OCH₃ H H CH₃ N N C C 64.1 ± 4.2 100~180 5767 H OCH₃ H CH₃ N N CC >180 >180 Pyrrole 5776 H Cl H H N C C C >180 >180 5775 Cl H Cl H N C CC >180 >180 Furan 5768 Cl H Cl H C O C C >180 >180 5769 Cl H Cl H C O CC >180 >180 5772 F H H H C O C C >180 >180 5770 H Cl H H C O CC >180 >180 5588 OCH₃ H H H C O C C >180 >180 5587 CH₃ H H H C O CC >180 >180 Imidazole 5760 H H H CH₃ C N C N >180 >180 5852 F H H H C NC N 34.3 ± 0.6 67 Thiazole 5583 H Cl H H C N C S >180 >180

Structures for certain of the capping groups of Table 7 are as follows:

By inference in comparing 5763 5764 and 5773, the above compound isbelieved to have increased activity compared to 5773 (i.e. <4 μM).

The above compound is believed to have an activity of <5 mM, inferredfrom the using the best triazole Ncap (above) and also combining theadditive effects of the 3 and 4 fluoro subsitutions on the3-phenoxy(pyridinyl-2-yl)methylamine system.

The series of heterocycles of Table 7 included incorporation of5-phenylfuran-2-carboxylic acid, 1-phenyl-1H-pyrrole-3-carboxylic acid,and 4-phenylthiazole-2-carboxylic acid in order to interrogatestructure-activity of the 5 membered ring. This enabled determination ofthe consequences of replacing the bridging atom between the phenyl andcarboxylic acid substitutions and also to probe the contribution of the5-methyl substituent in the pyrazole and triazole contexts.

Crystal structures of the 3,5 dichlorophenyl and the 4 chlorophenyltriazole were solved and provided insights into the protein-ligandinteractions of this N-terminal PLA. These included increasedcomplementarity of the 3-chloro with the secondary hydrophobic pocketrelative to the 4-chloro substitution, a hydrogen bond acceptor from the2-nitrogen of the 1,2,4-triazole to Trp217 NH and contribution of anadditional H-bond acceptor from the amide carbonyl to the carboxamideside chain of Gln254. In order to assess the contribution of the PLAH-bond acceptor, analogous compounds were made in each series andactivities for the 4-chloro and 3,5-dichloro analog were evaluated ineach context. The resulting SAR around the heterocyclic scaffoldsdetermined that while isosteric ring systems are presented, significantdifferences are observed in the in vitro potencies as measured in thefluorescence polarization (FP) binding assay.

The triazole containing N capping group was found to act as the bestscaffold, followed by the pyrazole, furan and triazole substructuresrespectively. These differences are manifest in In the CDK4/cyclin D1context, the fragment ligated inhibitory peptide that was capped withthe 4-chlorophenylpyrazole was found to be at least 4 fold less activerelative to the triazole N capping group. SCCP ID No. 5770, possessing afuran core structure, was found to be 25 fold less potent in this assay.The two pyrrole containing structures were found to be completelyinactive in the binding assays. Comparisons of the 3,5 substitutedphenylheterocyclic derivatives revealed a similar trend in binding tothe 4-chloro versions. With the relative potencies of the heterocyclicframework established, the versatility of the phenylpyrazole andphenylfuran carboxylic acid syntheses was exploited in order to generatemore diverse substitutions. In particular, 3-Cl, 3-F, 3 and 4 methoxy, 3methyl and unsubstituted phenyl rings were incorporated. Results fromthe pyrazole context suggested that beneficial substitutions include the3-Cl, 3-F and 3-H on the phenyl ring. The most potent compounds in thefuran isostere included the 3-Me which is 2 fold more potent than the4-Cl (the most active compound in this series). One imidazole(5-methyl-2-phenyl-2H-imidazole-4-carboxamide) and2-(4-chlorophenyl)thiazole-4-carboxamide were incorporated on to thepeptide, however these were found to possess little activity as Ncapping groups. Another imidazole derivative,2-(3-fluorophenyl)-1H-imidazole-4-carboxamide was synthesized, ligatedto the RLIF tetrapeptide to form a fragment ligated peptide, and foundto have similar activity to the pyrazole core structure.

A method for forming phenyl triazoles (e.g., SCCP ID Nos. 5843, 5773 and5774) is given in 3-steps below. The particular procedure is for thesynthesis of the N-cap for compound 5773, and a similar procedure may beutilized for other phenyl triazoles as will be evident to one ofordinary skill in the art:

Step 1. Procedure to make (E)-ethyl2-chloro-2-(2-(3,5-dichlorophenyl)hydrazono)acetate

-   1. Add 10 ml of 6N HCl to a solution of 3,5-dichloroaniline in 10 mL    of MeOH at 0 degree C.-   2. Sodium nitrite is added slowly-   3. Stir reaction for 15 minutes at 0 degree C.-   4. Sodium acetate is added to adjust the pH to 5-   5. A solution of ethyl 2-chloro-3-oxobutanoate (ethyl    2-chloroacetoacetate) in 10 ml of MeOH is added slowly at 0 degree    C.-   6. Bring to room temperature and stir the reaction for 12 hours-   7. Remove the MeOH under reduced pressure and add diethyl ether-   7. Separate and wash the organic layer with saturated sodium    bicarbonate and water-   8. Dry over sodium sulfate

Step 2. Procedure for ethyl5-methyl-1-(3,5-dichlorophenyl)-1H-1,2,4-triazole-3-carboxylate

-   1. Prd from step 1. and acetaldehyde oxime are dissolved in toluene    and heated to reflux-   2. Moniter rxn by TLC-   3. Once TLC shows consumption of half of the starting material,    additional 0.5 EQ of TEA is added and refulxing is continued-   4. Moniter rxn by TLC-   5. Rxn is concentrated and partitioned between EtOAc and H₂O-   6. Layers are separated and the aqueous layer is washed with EtOAc-   7. THe combined organics are washed with H₂O and brine-   8. Dry with Na2SO4 and filter-   9. Then concentrate-   10. Crystalize with Et2O (ethyl ether anhydrous) and hexane

Step 3. Procedure to make5-methyl-1-(3,5-dichlorophenyl)-1H-1,2,4-triazole-3-carboxylic acid

-   1. Add 13.3 mL of Ethanol and 13.3 mL of H₂O to product from Step 2.-   2. Reflux the contents for ˜2 hrs-   3. Evaporate the ethanol-   4. Add H₂O and extract with EtOAc-   5. Acidify with 1N HCl to ppt the prdt; if the prdt does not ppt,    extract aqueous layer with EtOAc 2-3 times, combine EtOAc wash and    wash with brine and dry with NA2SO4

Several additional analogs were synthesized in addition to the 3,5dichloro and 4-chlorophenyl analogs. In addition to determining theirinhibition of CDK2/cyclin A, the availability of the CDK4/cyclin D1binding assay was exploited to develop SAR for both kinases.Interestingly, the relative potencies of the 4-chloro and3,5-dichlorophenyl triazole (25 and 12 μM for CDK2/cylin A respectively)were reversed in the CDK4/cyclin D1 context (11 and 27 μM). This is theresult of different requirements of the secondary hydrophobic pocket inthe two cyclins as previously delineated through the study of thebinding of peptide analogues.

Validation was carried out to ensure that the method was efficient toproduce reproducible results and to show that the docking results of theunknown compounds were predictive. Two native ligands(1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carbonyl and1-(4-chlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carbonyl) and a negativecontrol were docked in both the sub units A and B of CDK2/Cyclin Acrystal structure. The variation in the parameters (i) energy grid(Dreiding, CFF and PLP1), (ii) minimization sphere (on or off) and (iii)number of poses generated (20, 10 and 5) was carried out.

For each parameter, the number of correct poses (poses that aresuperimposible with the crystal structure binding mode) of the positivecontrol ligands generated, the number of negative control poses in top25 poses, the best scoring functions that gave more number of correctposes in top ranking order were studied. The optimized parameters areenergy grid PLP1 with minimization sphere on and number of poses 10 andthe scoring function PLP1. The docking of the native ligands werereproducible with the optimized parameters. The results are shown inTable 8.

TABLE 8 Energy Grid Dreiding CFF PLP1 No. of correct poses 2 4 63,5-DCPT No. of correct poses 8 4-DCPT Negative controls in −PLP1(4),−PLP2(4), −PMF (7), DOCK No −ve control poses in top 25 Jain (4),PMF(4), SCORE (6) all the scoring functions DOCK SCORE(6) Best scoringLigScore2_Dreiding PLP1, PLP2 PLP1, PLP2 function 3,5-DCPT (rank of 4, 5(for all the PLP1 (9, 10, 11, 12, 25), PLP1 (7, 8, 9, 10, 14, top 25correct/closer scoring functions) PLP2 (13, 14, 15, 16, 25) 11, 12, 13,25), poses for the best PLP2 (11, 12, 13, 14, scoring function 18, 15,16, 17) 4-DCPT (rank of top PLP1 (1, 2, 3, 4, 5, 6, 7, 8), PLP1 (1, 2,3, 4, 5, 6) 25 correct/closer PLP2 (17, 18, 19, 20, 21, PLP2 (20, 21,22, 23, 24, 25) poses for the best 22, 23, 24) scoring function)

Molecules may be designed on the basis of one or more of: (i) Molecularweight less than 250, (ii) absence of charge on the molecules to improvepermeation, (iii) Presence of a carboxylic acid group which is essentialfor ligation to the peptide and (iv) Commercial availability andsynthetic feasibility.

Various N-capping group designs are shown below. The scheme fordevelopment of the designs generally includes:

-   1. Ring A is replaced with 5 membered or six membered heterocycles-   2. Ring B was replaced with phenyl group or heterocycles-   3. Spacer between two rings-   4. Spacers before the carbonyl group.

Other scaffold series were utilized as a basis for development of thesynthetic inhibitors in addition to the 1,2,4-triazole series discussedabove. Scaffolds included the following:

Examples of capping groups and calculated structure activity for each ofthe scaffold groups are further described in Table 9 (furans andthiazoles, Scaffold II), Table 10 (benzoic acids, Scaffold III), Table11 (picolinic acids, Scaffold IV), and Table 12 (phenyl acetic acids,Scaffold V), presented below.

TABLE 9 CDK2/ CDK4/ cyclin cyclin SCCP A IC₅₀ D1 IC₅₀ ID R₁ W X Y Z (mM)(mM) 5581 CH₂N (CH₂CCH₃)₂ C C C C 47.1 ± 19.2 40.7 5585 CH₂-imidazole OC C C 100~180 158.8 5586 CH₂-pyrazole O C C C 100~180 >180 5589 CH₂- O CC C >180 47.1 methylpiperazine  5761* CH₂- O C C C 100~180 >1804methylpyrazole  5582* 3-thienyl C N C S 70.7 ± 18.6 >180 5584 2-thienylC N C S >180 >180

As can be seen, the benzoic acid derivatives (Scaffold III, Table 10)gain more potency with substitutions on both the R₁ and R₂ positions ascompared to unsubstituted benzoic acid. At the R₁ position,substitutions such as meoxy and phenoxy improve the potency of thecompound by more than 2-fold, while at the R₂ position, the foursubstitutions introduced in Table 10 greatly enhance the activity by 26,9, 3, and 3 fold, respectively. In addition, the presence of basicgroups on the four structures suggests that the basic groups areimportant for binding to cyclin D.

TABLE 10 (Table 10 discloses “RLIF” as SEQ ID NO: 52, “RLNpfF” as SEQ IDNO: 54, “X-RLIF” as SEQ ID NO: 55 and “ALIF” as SEQ ID NO: 56) SCCP IC50(μm) ID R1 R2 Peptide link CDK2/A2 CDK4/D1 5857 H H RLIF   >100  100-180 5835 CH₃ H RLNpfF Lost ~200 (LIF) 5858 C₂H₆O H RLIF   >100   49,33.9 μm (0321) 5844

  C₅H₁₂O H RLIF   >100     >180   5846

  C₇H₈O H RLIF   >180     >100   5882

  C₃H₈0 H RLIF   >100     >100   5883

  C₄H₁₀O H RLIF   >100     >100   5851 H

  C₆H₁₃NO RLIF 32.8 ± 13.5 3.6 ± 0.28, 3.4 (0321) 5850 H

  C₅H₁₂N₂ RLIF 40.5 ± 9.8  11.5 ± 0.14, 11.6 (0321) 5566 H

  C₆H₁₄N₂ RLNpfF 18.2 ± 1.8  27.9 ± 2.97  541 H

  C₂H₇N₃ RLNpfF   6 ± 1.6 35.1 ± 4.10 5895 H

X-RLIF (peptoid) >>100   >>180   5896 OCH₃

RLIF 13.2 ± 3.7  12.3 ± 0.07 5919 H

  C₆H₁₄N₂ RLIF      16.69 5923 H

  C₆H₁₄N₂ ALIF   >100   (ALIF)     149.18 5920 OH

  C₆H₁₄N₂ RLIF      5.86 5922

  C₄H₁₀O

  C₆H₁₄N₂ RLIF      4.8       42.19 5921 H C₇H₁₆N₂ RLIF 6.04, 6.6 5965 H

Arg{βhomoLeu}NMePhe-NH₂     22.9      44.83 5966 OH

  C₆H₁₄N₂ Arg{βhomoLeu}NMePhe-NH₂      3.91      4.93 5968

  C₃H₈O

  C₆H₁₄N₂ Arg{βhomoLeu}NMePhe-NH₂      14.99      8.73 5967 H

  C₁₂H₂₅N₃ Arg{βhomoLeu}NMePhe-NH₂      10.03 5969 H

  C₇H₁₆N₂ Arg{βhomoLeu}NMePhe-NH₂      16.64      33.74 5970 H

  C₇H₁₆N₂ Arg{βhomoLeu}NMePhe-NH₂      4.91     297.37

As can be seen in Table 11, some of the Scaffold IV compounds show lessactivity as compared to unsubstituted picolinic acid, expect for thesubstitution of piperazine at the R₂ position.

TABLE 11 SCCP IC50 (μM) ID R₁ R₂ CDK2/A2 CDK4/D1  525 H H 39.3 ± 6.2/ 64(LIF), 94.3 ± 14.9 49 (LIF) (LIF) (0321) 5845 CH₃ H >100 100~180  524MeO H 70.1 ± 7.9 100~180  523 EtO H 47.5 ± 4.4/ 100~180 114 ± 10.5 (LIF)5856 H

~100 34.2

The Scaffold V compounds of Table 12 based on hydroxyphenyl acetic acidshow very good potency with two ethoxy substitutions on the 3 and 4positions of the phenyl ring. Moreover, as substitution at the R₁position gets bulkier, the potency of the Scaffold V compounds increase.While not wishing to be bound to any particular theory, the differencein potency between the 3-ethoxy and 3,4-diethoxy compounds may suggestthat the 3,4-diethoxy compound binds to cyclin D in a distinct mode.

TABLE 12 IC50 (uM) SCCP CDK2/ CDK4/ ID R₁ R₂ A2 D1 5854

H   >100 >100 5853

H   >100 ~100 5855

H >>100 10~100  530

6.5 ± 1.3/ 15.5 ± 3.3 (LIF) 24 (LIF)Arginine Isosteres

In one embodiment, isosteric arginine derivatives can be utilized aslinkers between 35DCPT and a C-terminal dipeptide,βHomoLeu-NMethylPhe-NH₂. These synthetic amino acids can form amidebonds similarly to natural amino acids but have side chains thatdistinguish them.

Examples of arginine isosteres can include those having the followingstructure:

In which the R group can be one of the following:

These synthetic amino acids can be synthesized and incorporated into theinhibitor compounds to enhance pharmacokinetics and inhibitoryactivities of peptides. The synthetic arginine derivatives obtained may

(1) potentially mimic the interactions that are made between the CBM(ARG5) and the CBG (ASP283),

(2) evaluate the steric requirements of this position,

(3) improve drug-like properties, and

(4) most likely improve the binding affinity of the compounds throughfavoring increased ion pairing interactions.

The replacement of Arg5 with various amino acids has been done withactivity still present however at a decreased amount of 4-fold orgreater. The arginine at this position is important for the bindingaffinity of HAKRRLIF (SEQ ID NO: 2) and therefore varying thefunctionality of these arginine derivative side chains can aid in thefurther development of the SAR and possibly the replacement for thisposition.

The arginine isosteres listed above were Fmoc-protected prior to solidphase peptide synthesis with a dipeptide C-cap, βHomoLeu-NMethylPhe-NH2.These compounds were then N-capped with 35DCPT. The alkylation of theguanidine group in these isosteres provided functionality and isbelieved to protect the guanidine groups from reacting during peptidesynthesis. These side chains are expected to interact with ASP283 of theCBG and help establish further SAR.

These synthetic arginine derivatives have been utilized to replace Arg5of the C-terminal pentapeptide (RRLIF). The side chains of thesearginine derivatives can contain functional groups such as acyclic(methyl and ethyl) and cyclic (4,5-dihydro-1Himidazole and1,4,5,6-tetrahydropyrimidine) groups. These groups may provideprotection to the guanidine group to elude reactivity during peptidesynthesis.

ADMET (absorption, distribution, metabolism, excretion, and toxicity)parameters were calculated for these arginine isosteres were calculatedand are described in Table 13, below.

TABLE 13 # Polar Isostere Molecular Solubility Absorption # # RotatableSurface structure Weight Level Level LogP HBD HBA Bonds Area

116.12 5 0 0.308 5 3 4 63.64

130.13 5 0 0.514 4 3 5 49.65

144.15 4 0 0.720 3 3 5 38.03

142.15 5 0 0.863 4 3 6 49.65

142.13 4 0 0.538 3 3 4 38.03

156.15 4 0 0.601 3 3 4 38.03

As can be seen, alteration of the arginine affects mainly log P and PSA.The solubility and absorption levels remain consistent with the alteredside chains and the number of HBD, HBA, and rotatable bonds arecomparable across the table. However, the PSA values decrease by 13 to25 units and the log P values increase with added functionality. Withthe increase in log P and the decrease in PSA, these arginine isosteresare expected to be promising replacements for natural arginine.

C-Terminal Partial Ligand Alternatives: Derivatives of1-phenyl-1H-1,2,4-triazole-3 carboxamide.

The computational enrichment strategy described herein was applied toidentify potential non-peptidic replacements for a C-terminalphenylalanine which has been verified as a critical determinant forbinding to the cyclin groove. The general structure of the C cappinggroups was as follows:

in which

-   -   R₅ is 4-chloro or 3,5-dichloro,    -   X is C, N    -   R₆, R₇, R₈, R₉ are independently H, CH₃, or halogen

One method for forming the C-capping groups is as follows:

Resin is swelled in CH₂Cl₂ for 30 min. Fmoc-Leu-OH and DIPEA are addedand the reaction allowed to stir for 2 hours. The solution is drainedand the resin treated 3× with CH₂Cl₂/MeOH/DIPEA 17:2:1 to cap unreactedchloride. The resin is washed with CH₂Cl₂ 3× and DMF 3×. The resin istreated with 20% piperidine in DMF for 30 minutes. The solution isdrained and the resin washed with CH₂Cl₂ 3× and DMF 3×. Fmoc-Arg(Pbf)-OHis dissolved in DMF along with HBTU and DIPEA. The solution is added tothe resin and allowed to stir for 3 hours. The solution is drained andthe resin is washed with CH₂Cl₂ 3× and DMF 3×. The resin is treated with20% piperidine in DMF for 30 minutes. The solution is drained and theresin washed with CH₂Cl₂ 3× and DMF 3×.1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carboxylic acid isdissolved in DMF along with HBTU and DIPEA. The solution is added to theresin and allowed to stir for 3 hours then sit overnight. The well isdrained and washed with CH₂Cl₂ 3× and DMF 3×. The resin is cleaved with5% TFA in CH₂Cl₂ and the crude is used as is after concentrating. Theprotected peptide2-((S)-2-(1-(3,5-dichlorophenyl)-5-methyl-1H-1,2,4-triazole-3-carboxamido)-5-((E)-2-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino) pentanamido)-4-methyl pentanoic acid is dissolved inmethylene chloride and appropriate amine, (e.g. 3-phenoxybenzylamineetc), HBTU and triethylamine are added. The solution is stirred untilHPLC indicates complete consumption of SM. The solution is concentratedand the residue partioned between EtOAc and water. The aqueous layer isback extracted with EtOAc and the combined organics are washed with 1NNaOH, 1N HCl, and brine. The organic is dried with NaSO₄, filtered andconcentrated. Purification is performed by semiprep HPLC if necessary.The product is then treated with 95:2.5:2.5 TFA:H2O:TIPS and allowed tostir overnight. The solvent is removed and the residue triturated inether. The solid is collected and purified by semiprep HPLC (Method: 0to 80 over 20 min).

Specific C capping groups examined are described further in Table 14,below.

TABLE 14 CDK2/ CDK4/ SCCP cyclin cyclin ID A IC₅₀ D1 IC₅₀ No. R5 X R6 R7R8 R9 (mM) (mM) 5807 4-chloro C H H H H 106.1 ± 26.2 >200 5824 4-chloroN H F H H  53.2 ± 11.6 >200 5823 4-chloro N H H F H 18.1 ± 4.0 >200 58224-chloro N H H Cl H 54.4 ± 0.9 >500 5825 4-chloro N CH₃ H H H  60 ±3.5 >500 5848 3,5- N H Cl H H   61 ± 10.8 120 dichloro 5849 3,5- C H ClH Cl >>180 120 dichloro

An SAR study of the bis aryl ether compounds revealed that3-phenoxybenzylamine was the most effective C-capping group although itsactivity relative to the native residue was diminished by approximately2 fold (Table 8). It has been shown in various studies of CGI peptidesthat incorporation of a halogen into the aromatic ring of Phederivatives results in substantial potency increase. Inclusion of 4-FPhe into the p21 CGI sequence resulted in a modest potency increasehowever 3-Cl Phe leads to a 10-20 fold enhancement in the CDK2/cyclin Acontext. Similar substitutions were incorporated into a3-phenoxy(pyridin-2-yl)methylamine system as a close structural analogueof the phenoxybenzylamine core structure. These included the 2-methyl, 3and 4-fluoro and 3 and 4-chloro substitutions of the phenoxy ringsystem.

Other C-caps have been identified and synthesized. In one embodiment aC-cap is 2-amino-N-ethyl-4-methyl-N-(3-phenylpropyl)pentanamide (C-capin SCCP 6005 and 6014; Table 15, below).

FIG. 7 illustrates this inhibitory group superimposed with HAKRRLIF (SEQID NO: 2) in the primary hydrophobic pocket of Cyclin A. From thisimage, it is evident that the phenyl propylamine group closely mimicsthe interactions of Phe8. The predicted binding mode of this groupsuggests that when incorporated as an inhibitor, this compound will aidin retaining the binding affinity of RRLIF in the primary hydrophobicpocket. The synthesis of this small molecule can be carried out via atwo-step process with good versatility and can allow facileincorporation of an N-cap and arginine residues. This fragment,2-amino-N-ethyl-4-methyl-N-(3-phenylpropyl)pentanamide, was synthesizedvia reductive amination followed by peptide synthesis as shown below andpurified through semi-preparative High Performance Liquid Chromatography(prep-HPLC).

Ccap Coupling

Peptide Synthesis

Initially in the formation scheme, the amine can undergo reductiveamination to form a secondary amine. Second, the N-capped peptide can bemade using solid phase synthesis. Next, the secondary amine can becoupled to the N-capped peptide in a solution phase coupling reaction.In the final step, the arginine side chain can be deprotected.

In addition to 2-amino-N-ethyl-4-methyl-N-(3-phenylpropyl)pentanamide,other variations have also been synthesized as shown in Table 15, below.The general structure of each of these inhibitors is as follows:

wherein

-   -   n1 is 1 or 0    -   n2 is 1, 2, or 3    -   the aromatic ring bonded to the (CH₂)_(n2) group can include one        or more halogens on the ring    -   R is hydrogen or ethyl.

TABLE 15 CDK2/Cyclin A CDK2/Cyclin D1 SCCP n1 n2 R IC₅₀(μM) IC₅₀(μM)6000 1 1 H >180     >180 6001 1 1 Et >180     >180 6002 1 2H >180     >180 6003 1 2 Et 85.62 >180 6004 0 3 H 57.74 >180 6005 0 3Et >180     >180 6014 0 3 Et 90.19 >180

Each of the inhibitors of Table 15 have been coupled to 35DCPT-Arg andeither Leu or Beta-Leu, the chemical structures of which are as follows:

The 35DCPT N-cap has been determined to be the most potent in thereplacement of the N-terminal. The variations in the table includedifferent length alkyl chains, and a hydrogen or ethyl group at the Rposition. The natural leucine has been used in the synthesis of theanalogs with a propyl alkyl chain (SCCP 6004 and 6005). Natural leucinehas the amine group on the carbon in the a position, to the carboxylicacid, which helps orient these C-caps in a binding mode similarly to thenative ligand, aids in the positioning of these fragments in the primaryhydrophobic pocket, and potentially aids in retaining the bindingaffinity of LIF. The non-natural βHomo-leucine was used in the synthesisof the analogs with either methylene or ethylene alkyl chains (SCCP6000, 6001, 6002, and 6003). Beta amino acids contain an additionalmethylene group between the carboxylic acid and the amine group wherethe extra carbon theoretically should adjust for the shorter alkylchains and help position the fragments in the primary hydrophobic pocketto closely mimic the native ligand. Following peptide synthesis (ofN-cap-Arg-Leu/βLeu), coupling with the C-cap and arginine deprotection,these compounds were purified through prep-HPLC.

The compounds were found to be 88-98 percent pure, by UV, and the MSresults confirmed the identity of the desired compound. Once thecompounds were characterized, their binding affinities were tested by FPassay. The competitive binding assay determined that the ethylphenylethan-amine C-cap coupled to 35DCPT-R-βHomoL (SCCP 6003) has anIC50 value of 85.62 μM and the phenylpropylamine and ethyl phenylpropylamine C-caps, coupled with 35DCPT-RL (SCCP 6004 and 6014 respectively),had an IC50 values of 57.74 μM, and 90.19 μM against CDK2/cyclin A,respectively. All other compounds in the table have IC50 values greaterthan 180 μM against CDK2/cyclin A.

Table 16, below, presents additional embodiments of inhibitors as havebeen developed by use of the methods described herein. Prostate (DU145)and osteosarcoma (U2OS) cell lines were treated with SCCP 5963 and 5964,with results presented in Table 16. These particular inhibitors have thegeneral structure of:

TABLE 16 Competitive Binding Assay CDK2/ CDK4/ Cell Proliferation AssayCyclin A Cyclin D1 DU 145 U2OS SCCP R1 R2 IC50 (μM) IC50 (μM) IC50 (μM)IC50 (μM) 5963 Me

6.49 ± 0.27 >100 36.5 ± 2.6  30.7 ± 2.7 5964 H

7.91 ± 5.08 >100 21.8 ± 0.32 18.3 ± 1.5

ADMET (absorption, distribution, metabolism, excretion, and toxicity)parameters were calculated for SCCP 5963, 5964, and 6004 and aredescribed in Table 17. ADMET parameters such as solubility between 0-1and absorption between 3-4, Lipinski's Rule of 5 which states that themolecular weight (MW) for oral bioavailable drugs should be <500, logP<5, the number of hydrogen bond donors (HBD)<5, and the number ofhydrogen bond acceptors (HBA)<10 and the number of rotatable bonds (≤10)and polar surface area (PSA) (≤140) are ideal for cell permeability.SCCP 5964 was the most active in the cell proliferation assay and whencompared to SCCP 5963, it was found to have a more ideal solubilitylevel, lowest log P value, fewer HBA and rotatable bonds but has thehighest PSA. The most active compound from the C-caps was SCCP 6004.This compound has comparable solubility and absorption levels and hasfewer HBD, HBA, and rotatable bonds. The log P value for SCCP 6004 wassignificantly higher than that of SCCP 5964 but has almost 1.5 fold lessPSA. This may conclude that SCCP 6004 can have similar cell permeabilityand activity as SCCP 5964. With these ADMET values it can be predictedthat SCCP 6004 can have greater cell permeability but may have slightlyless activity based on its binding affinity in the FP assay.

TABLE 17 # Polar Molecular Solubility Absorption # # Rotatable SurfaceSCCP Weight Level Level LogP HBD HBA Bonds Area 5963 714.292 2 3 3.163 814 18 214.21 5964 706.233 1 3 2.255 9 14 18 251.25 6004 658.279 2 34.044 8 12 17 181.65

Table 18, below, presents another series of C-caps as may beincorporated in an inhibitor. These C-caps can be synthesized accordingto a scheme as provided above for the materials of Table 15. The C-capsof Table 18 differ from SCCP 6000-6005 and 6014 by the group in the Leu6position which is either leucine or βHomo-leucine. These C-caps cancontain phenylalanine, histidine, βHomophenylalanine, or βHomo-histidinedepending on the length of the alkyl chain at the n2 position. Thesederivatives can help further establish the SAR for the Leu6 position.The variation of the ring size plus the addition of potentialion-pairing interaction of the imidazole ring of histidine can aid inthe determination of which functional group should be present to retainor enhance the binding affinity of the Leu6 position. Leu6 haspreviously been determined to be the most critical amino acid of theoctamer (HAKRRLIF (SEQ ID NO: 2)) and therefore its replacement can bechallenging but has also been shown that this position is capable to bereplaced with a small molecule. The inhibitors of Table 18 have thestructure:

-   -   wherein        -   n1 is 0 or 1,        -   n2 is 3,        -   R1 is an arginine side chain,        -   R2 has one of the following structures:        -   R2 has one of the following structures:

-   -   -   R3, R4, and R5 are independently hydrogen or a halogen.

TABLE 18 Sample no. n1 n2 R2 R3 R4 R5 1 2 0 1 3 3

H H H H F F 3 4 0 1 3 3

H H H H F F 5 6 7 8 0 0 0 0 3 3 3 3

Cl H F H H Cl H F Cl H F H

Table 19, below, presents the ADMET parameters for these compounds.

TABLE 19 # Polar Sample Molecular Solubility Absorption # # RotatableSurface no. Weight Level Level LogP HBD HBA Bonds Area 1 706.279 2 34.510 8 12 18 181.65 2 692.263 2 3 4.054 8 12 17 181.65 3 682.254 2 32.398 9 14 17 210.33 4 696.64 2 3 2.083 9 14 17 210.33 5 726.201 1 35.373 8 12 17 181.65 6 692.240 1 3 4.708 8 12 17 181.65 7 694.260 1 34.455 8 12 17 181.65 8 676.269 2 3 4.250 8 12 17 181.65

These compounds have comparable solubility and absorption levels.Compound No. 4 has lower log P and PSA values than SCCP 5964, which mayindicate increased cell permeability and activity. Compound No. 3 has acomparable log P value but a lower PSA value which may result in thiscompound having comparable permeability and activity as SCCP 5964. Allother compounds have greater log P values, similar number of HBD, HBAand rotatable bonds, and lower PSA values. This may contend that thesecompounds may have cell permeability and activity but will most likelynot be greater than that of SCCP 5964.

Following development of C-terminal and N-terminal groups, certainoptimized terminal groups can be combined into individual molecules.While the unsubstituted bis-aryl ether (incorporating3-phenoxybenzylamine) had decreased activity when combined with the3,5-DCPT-Arg-Leu N-terminal group relative to the previous peptidecontext (Arg-Arg-Leu-3PBA), addition of halogen substituents onto thearomatic ring contacting the primary lipophilic site resulted inrecovery of binding and comparable activity to the native peptidesequence. Individually, a 3-fluoro and 4-fluoro substituted bis-arylether had enhanced potency compared to the unsubstituted. Addition ofthese halogens follows a similar pattern to that observed in the peptidecontext where incorporation of either a 3 or 4 substituted phenylalanineresidue resulted in significant potency gains. These results illustratethat removal of peptide determinants and substitution with fragment likecompounds can change the binding mode of an inhibitor and result inpotency loss. The data obtained also suggests that reoptimizationthrough SAR studies can regain potency lost in this way and that moredrug-like and less peptidic inhibitors can be obtained.

Examples of such molecules include the following cyclin A selectivecompounds:

and the following cyclin D selective compounds:

In one embodiment, picolinamide and benzamide N-cap scaffolds can beused for potential small molecules to replace HAKRRLIF (SEQ ID NO: 2) orto aid in the design of novel C-caps. The validated optimized parameterscan be used in the docking of the resulting compounds. One inhibitordeveloped from this approach isN-(5-guanidino-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl)benzamide (FIG.6), which contains a benzamide N-cap, a naphthalene C-cap, and anarginine in the Arg5 position.

The benzamide Ncap, of this small molecule, nicely forms an H-bond withTrp217 and the arginine ion-pairs well with Asp283 and H-bonds withGlu254. In addition, the naphthalene Ccap has good complementarity withthe primary hydrophobic pocket. This compound also ranks well in theoverall scoring functions. LigScore2_Dreiding, -PLP1, and -PLP2 havebeen determined to be good scoring functions for the prediction ofpotent small molecules. This compound is ranked within the top twentyfive percent of all ligands docked with scores of 5, 56.06, and 55.88for LigScore2_Dreiding, -PLP1, and -PLP2, respectively. With theobserved intermolecular interaction and the resulting scoring functions,this molecule was expected to have activity against both Cyclin A andD1. However, upon testing in the FP assay this fragment was found to beweakly binding.

Modeling the Interactions of p27 with Cyclin D1 Structures

CDK4/cyclin D1 have been shown to associate with p27 and that thisinteraction promotes the formation of the complex. It is also known thatdifferent states of the ternary complex exist, where p27 may bind togenerate inhibited and non-inhibited CDK4 species. A critical aspect ofthis process is the phosphorylation of p27 on Y88, sited on the 3₁₀helix which inserts into the ATP binding site of CDK4 in the inhibitedcomplex. Phosphorylation presumably leads to dissociation of the helixfrom the ATP cleft through disruption of hinge H-bonding interactionsand through repulsion of the phosphate with nearby acidic residues. Inthis non-inhibited form, p27 however, must still maintain affinity forthe complex in order to sequester the inhibitor from CDK2/cyclin Ecomplexes and allow cell cycle progression. A major contribution to thisbinding is through cyclin D1/p27 interactions and assisted by the CBMand other residues. So as to construct a model structure of p27/cyclinD1 interactions, cyclin D1 isolated from the 2W96 crystal structure wasoverlayed with the CDK2/cyclin A/p27 ternary complex (1JSU). Afterdeletion of the CDK2, cyclin A and non cyclin D1 interacting p27residues, the newly formed complex was subjected to energy minimization.After convergence of the structure to a suitable minimum, andexamination of the resulting interactions, a plausible structural basisfor the interactions of p27 with cyclin D1 interactions was described.Subsequent to generation of this structure, the interaction energies ofindividual p27 residues with cyclin D1 were generated and compared withthose for cyclin A. Significant differences in the intermolecularinteractions are apparent for several residues, several of which arenoted in the octapeptide complexes described in the above sections.These include, A28, N31, F33, V36, L41 and L45. Comparison of themolecular surface for the p27 interacting residues of cyclin A vs. thoseof cyclin D1 indicated that profound differences exist specifically inthe region where the C-terminus of the inhibitory protein exits from theprimary hydrophobic pocket. The more extensive cleft of cyclin D1, ledto the hypothesis that incorporation of a suitable residue C-terminal tothe glycine would lead to preferential binding vs. cyclin A.Computational design of a number of different residues suggested thatmethionine would be a good candidate for more optimal interactions andtherefore synthesis and testing of the p27 sequences shown in Table 5,was completed and confirmed this conclusion.

FIG. 5 illustrates a modeled complex of p27 residues 25-49 with CyclinD1 (2W96) overlayed with SAKRNLFGM (SEQ ID NO: 1). The P35 and V36interacting site on cyclin D1 is the region shown to provide a moreextensive hydrophobic pocket than in the cyclin A2 context and which wasexploited by methionine substitution. As may be seen in FIG. 5, the P35and V36 contacting site of cyclin D1 has a larger accessible volume andtherefore has suboptimal interactions with p27. This was confirmed inthe per residue interaction energy calculation which yielded values of−1.9 and -3.6 kcal/mol for D1 and A respectively. The lack of increaseof the Asparagine containing sequence may be explained by the formationof an intramolecular H-bond observed in the crystal structure and whichprecludes optimal interactions of the methionine. Substitution of thisresidue with an alanine resulted in a 2 fold potency enhancement aspredicted. As illustrated (FIG. 5), the linear side chain of the P35Manalog extends with a high degree of complementarity into the extensionof the primary hydrophobic pocket. These results suggest that thisextended binding site in cyclin D1 could be exploited in the design ofsmall molecule cyclin groove inhibitors.

LEU-PHE Mimetics

Based on commercially available compounds with appropriate functionalityto mimic both Leu6 and Phe8 and by use of the disclosed methods,additional compounds for use as C-caps as described below in Table 20were designed, synthesized and coupled to 35DCPT-Arg5. These compoundscan have the general structure:

-   -   wherein        -   n is 0 or 1        -   R1, R2, and R3 are independently hydrogen, isobutyl, methyl,            ethyl, or propyl groups.

These Leu-Phe mimetics contain either isobutyl or isopropyl groups atthe R2 position to help retain the interactions of Leu6 in the primaryhydrophobic pocket shown to be the most important residue of the CBM.Also, there are varying alkyl groups at the R1 position to mimic Phe8 inthe pocket. The general structure of the C-caps of Table 20 is asfollows:

TABLE 20 CDK2/ CDK2/ Cyclin Cyclin A IC₅₀ A IC0₅₀ SCCP n R1 R2 R3 (μM)(μM) 5977 0 iBut iBut H 148.4 >180 5978 0 iBut iBut H >180 >180 5979 0Pr iBut H 181.2 >180 5980 0 Pr iBut Me >180 >180 5981 1 Me iButH >180 >180 5982 1 Me iBut H >180 >180 5983 0 Et iBut H 164.02 >180 59840 Et iBut H >180 >180 5985 0 Me iBut H >180 >180

Following solid phase synthesis and coupling with the C-cap, the Leu-Phemimetics were purified via prep-HPLC. As these compounds have a chiralcenter and were purchased as a racemic mixture, diastereomers wereformed during synthesis. However, not all of the isomers were able to besuccessfully separated by prep-HPLC but the isomers that were separatedthe isomers that eluted first were those with activity. The isomers withactivity would be the R isomer, as the S isomer would not be expected tohave activity against the CDK/cyclin complexes because only S aminoacids have been shown to be active. The compounds were 79-98 percentpure, by UV and the MS results the identity of desired compounds wereconfirmed. Subsequent to full characterization of the compounds theirbinding affinities were tested via FP assay.

Of the compounds listed in Table 20, only SCCP 5977, 5979 and 5983showed activity against CDK2/cyclin A (FIG. 8) while none displayedsignificant binding to CDK4/cyclin D1. SCCP 5977 showed an IC50 value of148.4 μM, SCCP 5979 had an IC50 value of 181.2 μM, and SCCP 5983 had anIC50 value of 164.02 μM all against CDK2/cyclin A. These compounds werenot active against CDK4/cyclin D1. The R group for each of thesecompounds was 35DCPT-Arg.

The isobutyl group mimicking Leu6, in all the compounds, retained someof the binding affinity and the various alkoxy groups mimicking Phe8 hada significant effect on the activity of each compound on CDK2/cyclin A.SCCP 5977 contains an isobutyl group to mimic the Phe8 position in theprimary pocket. This compound had an IC50 value of 148.4 μM againstCDK2/cyclin A. SCCP 5979 has a propyl group reaching in to the pocket inreplacement of Phe8 and has an IC50 value of 181.2 μM againstCDK2/cyclin A. Lastly, SCCP 5983 contains an ethyl group which showed tobe slightly less potent against CDK2/cyclin A with an IC50 value of164.02 μM. All other compounds (SCCP 5978, 5980-5982, 5984, and 5985)were not active against either CDK2/cyclin A or CDK4/cyclin D1.

The isobutyl group replacement for Phe8 (SCCP 5977) appeared to be agood alkyl group compared to the propyl group (SCCP5979) and the ethylgroup (SCCP 5983). The additional methyl group of the isobutyl seemed tobe a contributor to the retention of potency compared to the propylgroup. SCCP 5977 showed an IC50 value of 148.4 μM, SCCP 5979 with 181.2μM and SCCP 5983 with 164.02 μM. The isobutyl side chain (SCCP 5977)proved to be most potent against CDK2/cyclin A with an IC50 value of148.4 μM.

In summation, comparison of the cyclin binding grooves of cyclin D1structures obtained recently through crystallographic studies providesconsiderable insight into the structural requirements for cyclin A2 vs.D1 selectivity and for differential binding of CGI peptide analogues.While the binding of peptide inhibitors of cyclin A and E substraterecruitment has been extensively characterized, little information hasbeen made available describing the determinants of cyclin D inhibition.Structural analysis revealed that two key amino acid substitutions inthe cyclin D1 groove have a major impact on peptide inhibitor binding.Exchange of one of the two acidic residues interacting with Arg4 (Asp216and Glu220), with Thr62 significantly decreases the calculated enthalpiccontribution to binding and is suggestive of a large decrease inaffinity. In order to determine if the predicted decrease in theelectrostatic interaction energy is significant in contributing tocyclin A selectivity, the arginine isostere citrullene was incorporatedinto the p21 8mer, HAKCitRLIF (Table 5) (SEQ ID NO: 26). It waspredicted that due to the less acidic environment of the Arg contactingresidues in cyclin D, that the potency decrease would be less marked inthis context. In reality however, a similar drop off was demonstrated inboth scenarios and thus indicating otherwise. Closer examination of thepeptide-cyclin D1 structure suggests that the urea carbonyl ofcitrullene is within H-bonding distance of the OH group of Thr62. Thisinteraction would therefore compensate for the decreased capacity to ionpair and result in a similar potency decrease.

As described, the second major difference between the two cyclins is inthe exchange of Leu214 in cyclin A for Val60 in cyclin D. The smallerValine sidechain projects down toward the base of this hydrophobicpocket with the net effect that the y methyls are brought into closerproximity to the peptide inhibitor side chains which insert into thispocket. This substitution therefore decreases the volume of the primaryhydrophobic pocket in the latter and thereby results in lower affinityof CGI peptides containing phenylalanine. Cyclin bound complexes weregenerated for a series of peptides previously determined to have varyingaffinities for cyclin A and cyclin D1 and possessing differentC-terminal sequences. The calculated binding energies for thesecomplexes correlated well not only for IC50s determined for cyclin A andD1 individually but also for the selectivity of the peptides observed.These results therefore determined that in addition to the X-raystructures used, the model structures for the peptide-cyclin complexesgave valid results and that this information is useful in the potentialdesign and optimization of improved cyclin D1 inhibitors. From theseobservations, the hypothesis was proposed that due to the decreasedvolume of the primary hydrophobic pocket relative to cyclin A, that theincorporation of non-natural amino acids with differing cyclicsidechains than phenylalanine might be tolerated to a greater degree. Tothis end, the results presented confirm that this is indeed the casehowever these are dependent on the peptide context. As has beenpreviously structurally characterized, the presence of a spacer residuebetween the critical Leu and Phe functions to allow a geometricalarrangement of the two side chains that interacts with a greater degreeof complementarity and therefore increases binding affinity relative topeptides with no spacer. The results suggest that non-spacer containingpeptide, SAKRRLXG (SEQ ID NO: 3), has a binding mode which is moreconducive and tolerant of smaller cyclic sidechains. In order to probethis further, a 3D structure for each of the synthesized analogs incomplex with both cyclins was generated and further to this, theirnon-bonded interaction energy calculated. These results suggested that acorrelation between the observed potencies and the calculated affinityexisted and confirmed that for both 5 membered rings, a decrease inbinding of these analogues would be expected. The structural basis forthe greater affinity of the furylalanine (X1) vs. the 2-thienylalanine(X2) in the p107 context is apparent from the modeled structure. Thecloser proximity of the heteroatom to Val60 in the peptide without thespacer residue results in displacement of the larger sulfur containingPhe replacement (thiophene ring) and lower relative affinity. In the p21peptide, the conformational preference allowed by the spacer residue,results in the heteroatom pointing to the back wall of the primaryhydrophobic pocket, away from Val60. As the heteroatom projects intomore expansive region, the larger sulfur atom provides greatercomplementarity with K96 and Q100 resulting in increased affinity in thethienylalanine derivative. Changing the context of the heterocyclicsulfur atom as in X3 resulted in potency increase of SAKRRLX3G (SEQ IDNO: 11) for cyclin A but an increase in cyclin D1 affinity. The largerhydrophobic pocket in cyclin A may accommodate the bulky sulfur atommore readily than may the cyclin D1 site decreased in volume by Val60.Examination of the intermolecular contacts for the cyclohexylalaninederivative X6, a bulkier Phe replacement as a result of the unsaturatedring, again provided insight into the differing potencies for peptidescontaining this residue with cyclin D1. Modeling of the complex ofSAKRRLX6G (SEQ ID NO: 14) with cyclin D1 (12 fold decrease in IC50),suggested that in order to maintain productive binding, the CHAsidechain is brought in close proximity to Val60 resulting inunfavorable contacts. For the HAKRRLIX6 (SEQ ID NO: 22) inhibitor (4fold loss in potency), the sidechain may adopt a more favorableposition, contacting several residues of the primary binding site inline with its higher relative potency. The dramatic decreases ininhibition of the pyridylalanine derivatives X7 and X8 relative to thenative phenylalanine cannot readily explained in terms of differentinteractions with the cyclin groove. A probable scenario is that thepyridyl ring is solvated to a greater degree relative to the phenyl andtherefore a desolvation penalty would disfavor binding. A number ofsubstitutions in the cyclin groove recognition motif have beenincorporated in the N-terminal and arginine binding site and provideadditional information on the tolerance of sequence changes upon bindingto the secondary hydrophobic and acidic regions of cyclin D1.

Disclosed methods can provide a plurality of benefits as compared toconventional approaches that are used for fragment based design in drugdevelopment. Firstly as potential fragment alternatives are evaluatedwhile ligated to truncated peptide sequences, a successful hit in thedisclosed methods provides a fragment ligated inhibitor thatrecapitulates binding of the intact native peptide. The truncatedpeptide therefore acts as an affinity scaffold and obviates the need fora highly sensitive detection method. This stands in contrast toconventional fragment based design that typically requires methods fordetecting milimolar binding affinity. Another requirement of fragmentbased design utilizing crystallography as a detection method is thenecessity for highly soluble fragments since by definition they musthave much higher solubility than their binding constant. The presentmethods can evaluate fragments while ligated to a peptide and thereforecan provide solubility through the polarity of the peptide sequence.Furthermore optimization of PLAs can be performed while in the fragmentligated inhibitor context of the disclosed method therefore againavoiding requirement for expensive and difficult methods for bindingdetermination.

While the subject matter has been described in detail with respect tothe specific embodiments thereof, it will be appreciated that thoseskilled in the art, upon attaining an understanding of the foregoing,may readily conceive of alterations to, variations of, and equivalentsto these embodiments. Accordingly, the scope of the present disclosureshould be assessed as that of the appended claims and any equivalentsthereto.

What is claimed is:
 1. A synthetic CDK/cyclin inhibitor that inhibitsinteraction of a complex formed between a first CDK protein and a firstcyclin protein with a substrate of the complex, the synthetic CDK/cyclininhibitor comprising a peptide sequence and a capping group bonded tothe N-terminus of the peptide sequence, the capping group comprising asubstituted benzoic acid, the peptide sequence comprisingArg-βhomoLeu-3-thienylalanine, the capping group having the followingstructure:


2. The synthetic CDK/cyclin inhibitor of claim 1, further comprising acapping group bonded to the C-terminus of the peptide sequence.
 3. Thesynthetic CDK/cyclin inhibitor of claim 2, wherein the capping groupbonded to the C-terminus is2-amino-N-ethyl-4-methyl-N-(3-phenylpropyl)pentanam ide.
 4. Thesynthetic CDK/cyclin inhibitor of claim 2, wherein the capping groupbonded to the C-terminus is an ethyl phenylethan-amine, aphenylpropylamine, or an ethyl phenylpropyl amine.
 5. The syntheticCDK/cyclin inhibitor of claim 2, wherein the capping group bonded to theC-terminus includes phenylalanine, histidine, βHomophenylalanine, orβHomo-histidine.